DNA is a highly dynamic biopolymer that undergoes sequence-specific structural changes in response to cellular trigger factors that are essential for fundamental processes such as replication, transcription, recombination, and DNA repair. The mechanism by which DNA undergoes functionally optimized conformational changes remains poorly understood. There is growing evidence that intrinsic sequence-specific flexibility guides DNA structural transitions along functional pathways; however, a direct test of this hypothesis has been hindered by lack of techniques that can be used to visualize DNA deformability at the atomic scale. Intrinsic flexibility also guides the DNA dynamic response to cellular supercoiling and bending forces. Despite growing evidence that such forces can dramatically affect DNA structure and function, the current DNA structure-function paradigm is based almost exclusively on studies of DNA in the more experimentally accessible relaxed duplex form. The goal of this proposal is to develop NMR methods, complemented by molecular dynamics simulations and biochemical assays, to visualize sequence and damage-specific DNA flexibility at the atomic scale in the presence and absence of supercoiling. Specific Aim 1 will test the hypothesis that DNA undergoes sequence-specific and spatially non-random thermally-induced fluctuations and that trigger factors, such as proteins, take advantage of this flexibility and induce specific changes in DNA structure by capturing distinct conformations from a pre-existing dynamical ensemble. These studies will focus on variable length A-tracts, dinucleotide CpA steps, their combination, and will explore the biological significance of sequence-specific flexibility in adaptive recognition. Specific Aim 2 will test the hypothesis that DNA damage induction is correlated to sequence-specific flexibility and that repair enzymes exploit the modified flexibility of damaged DNA and capture transient states from a dynamical ensemble rather than induce new ones by induced fit. These studies will focus on damaged DNA substrates of the base pair excision pathway enzyme human alkyladenine DNA glycosylase. Specific Aim 3 will develop minicircles as a model NMR system for experimentally characterizing DNA structural dynamics at atomic resolution in the presence of supercoiling. We will test the hypothesis that supercoiling dramatically affects the basic structural and dynamical properties of DNA, causing an increase in motional correlations between residues, promoting B-to-Z transitions, and enhancing the conformational deformability of A-tracts and damaged DNA, thus providing a mechanism for long-range signaling and communication. PUBLIC HEALTH RELEVANCE: There is growing evidence that sequence-specific DNA flexibility plays a fundamental role in key genetic transactions such as replication, transcription, recombination, and DNA repair that lead to pathology when improperly functioning. Understanding sequence-specific DNA flexibility is also of key importance for rationally designing small molecules that specifically bind to DNA and thus act as therapeutics or chemical tools for investigating diverse biological questions.